Methods for improving competency of corn plant immature embryo

ABSTRACT

The present invention provides methods for improving competency of plant cells, such as corn plant immature embryo for bacterial-mediated transformation comprising contacting the plant cells, such as corn plant immature embryo with an effective amount of polyethylene glycol (PEG) for a period of time prior to transformation. The ability to store and maintain competent plant cells, such as corn plant immature embryo for transformation and tissue culture allows more efficient planning and execution of large-scale experiments by providing flexibility of peak production hours, or during unplanned disruptions in the production process. These methods are useful in preserving the viability of plant cells, such as corn plant immature embryo in various storage conditions, thus improving their competency for transformation and tissue culture.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. Ser. No. 13/328,222, filedDec. 16, 2011, now U.S. Pat. No. 10,407,685, which claims the benefit ofpriority to U.S. Provisional Application Ser. No. 61/424,136, filed onDec. 17, 2010. The entire disclosures of the above application areincorporated herein by reference in their entireties.

FIELD OF THE INVENTION

This invention is in the field of plant biotechnology. Morespecifically, a method of improving competency of plants cells forbacterial-mediated transformation is provided.

BACKGROUND OF THE INVENTION

Plant tissues such as embryos can be obtained in large quantities bymechanical means, as disclosed in U.S. Pat. No. 7,427,695, for use intissue culture. Typically, once such plant tissues are obtained, theyare used immediately or within hours in tissue culture processes with orwithout a transformation step. When stored for later use, its competencyfor transformation may be reduced. Embryonic axes derived from dryseeds, for example as disclosed in U.S. Patent Application PublicationNo. 2008/0280361, are relatively stable upon storage, but a hydrationstep prior to transformation may reduce their competency fortransformation.

SUMMARY OF THE INVENTION

The present invention provides methods for improving competency of plantcells for bacterial-mediated transformation comprising contacting theplant cells with an effective amount of polyethylene glycol prior totransformation.

In certain embodiments, the effective amount of polyethylene glycol maybe from about 1% to about 25% by volume. In more particular embodiments,the effective amount of polyethylene glycol is about 20% by volume.

The molecular weight of the polyethylene glycol may range from about 200to about 10000, while in more particular embodiments the molecularweight of the polyethylene glycol is from 4000 to 8000.

In certain aspects, the composition further comprises an effectiveamount of at least one growth regulator, wherein the growth regulatorcomprises an amount of auxin, cytokinin, or combination thereof. Auxinsmay include IAA, 2,4-D, NAA, IBA, dicamba, or a combination thereof, andthe amount of auxin is preferably from about 0.001 mg/L to about 30mg/L.

The cytokinins may comprise BAP, zeatin, kinetin, TDZ, or a combinationthereof, and may be present in an amount from about 0.001 mg/L to about30 mg/L.

In practicing the present invention, the period of time for contactingthe plant cells with PEG may be from 1 day to about 30 days. In othermore specific embodiments, the effective period of time is about 3 daysto 7 days. In still other embodiments, plant cells are contacted withthe PEG for a period of about 30 min to 300 min.

The methods of present invention may further comprise a rinse step,wherein the plant cells that have been contacted with the PEG are rinsedwith a non-PEG containing composition prior to transformation.

Another aspect of the present invention further comprises regeneratingthe plant cells. The plant cells may comprise cells of seed, leaf, stem,root, immature embryo, mature embryo, callus, microspore, meristem,cotyledon, hypocotyl, epicotyl, mesocotyl, coleoptiles, radical, plumuleor reproductive tissue from a monocotyledenous or dicotyledenous plantspecies.

In practicing the methods of the present invention, the bacterialmediated transformation may be Agrobacterium-mediated, orRhizobium-mediated transformation.

A transgenic plant, and any plant parts thereof as defined elsewhere inthe present disclosure, created using the methods of the presentinvention disclosed herein, is also an aspect of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and form a part ofthe specification, illustrate the embodiments of the present inventionand together with the description, serve to explain the principles ofthe invention. In the drawings:

FIG. 1 illustrates callus production from embryos that were stored inLynx 2304 medium supplemented with PEG (left panel), compared to embryosthat were stored in Lynx 2304 without PEG (right panel).

FIG. 2 illustrates enhanced trichome formation and swelling of thecoleoptilar node from the embryos that were stored in Lynx 2304 mediumsupplemented with PEG and BAP (right panel) compared to the embryos thatwere stored without BAP (left panel).

FIG. 3 illustrates improved shoot formation from the embryos that werestored in Lynx 2304 medium supplemented with PEG and BAP (right panel)compared to the embryos that were stored in Lynx 2304 medium without BAP(left panel).

DETAILED DESCRIPTION OF THE INVENTION

The following is a detailed description of the invention provided to aidthose skilled in the art in practicing the present invention. Those ofordinary skill in the art may make modifications and variations in theembodiments described herein without departing from the spirit or scopeof the present invention. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs. Generally, the nomenclature and descriptions of basicmanufacture or laboratory procedures described herein are known andcommonly employed in the art. Unless otherwise noted, conventionalmethods are used for these procedures, and are exemplified by a varietyof general technical dictionaries or texts. Where a term is provided inthe singular, the inventors also contemplate aspects of the inventiondescribed by the plural of that term. The inventors do not intend to belimited to a particular mechanism or mode of action. Reference theretois provided for illustrative purposes only. All publications, patentapplications, patents, and other references mentioned herein areexpressly incorporated by reference in their entirety.

The present invention provides methods for improving competency of plantcells for bacterial-mediated transformation comprising contacting(applying, treating, storing, touching, joining, mixing, or to otherwisecause interaction) the plant cells with an effective amount ofpolyethylene glycol (PEG) for a period of time prior to transformationsufficient to enhance transformation of the so-treated tissue.

The methods of the present invention are useful in preserving theviability of plant cells in corn immature embryos during various storageconditions, thus improving their competency for transformation andtissue culture compared with tissues that are not contacted with aneffective amount of PEG. Hence, an “effective amount” of thecomposition, is defined as an amount that is efficacious for improvingcompetency for bacterial-mediated transformation of plant cells that arecontacted by the composition, as compared to plant cells that arecontacted by either none, too little, or too much of the composition soas to either be ineffectual, or detrimental to the plant cells'competency.

The ability to store and maintain competent plant cells fortransformation and tissue culture allows more efficient planning andexecution of large-scale experiments by providing flexibility of peakproduction hours, or during unplanned disruptions in the productionprocess. This also enables greater flexibility for shipping tissues todifferent sites. Also, such methods are useful in improving competencyof plant cells in embryonic axes from dry seed, for example, of soybeanfor transformation.

In order to provide a clear and consistent understanding of thespecification and the claims, including the scope given to such terms,the following definitions are provided.

The term “competency” is defined as a response of a plant cell to tissueculture or transformation. A “response” is typically defined inbiological systems as any behavior or change of a living organism thatresults from an external or internal stimulus. Hence, the term “improvedcompetency” is defined as enhancement over a basal or negative responseto tissue culture or transformation. In some aspects, the improvedcompetency to tissue culture may be measured by a higher rate ofsurvivability of the plant cells. In other aspects, improved competencyto transformation is measured as an increase in transformationfrequency.

The term “plant cells” generally refers to any cells from any part of aplant, including but not limited to seed, leaf, stem, root, immatureembryo, mature embryo, callus, microspore, meristem, cotyledon,hypocotyl, epicotyl, mesocotyl, coleoptiles, radical, plumule or flowercells including sepal, petal, stamen, pollen, pollen tube, pistil,receptacle, and ovule, among others. The plant cells may be derived fromessentially any plant, including but not limited to barley, corn, oat,rice, rye, sorghum, turf grass, sugarcane, wheat, alfalfa, banana,broccoli, bean, cabbage, canola, carrot, cassava, cauliflower, Chinesecabbage, celery, citrus, clover, coconut, coffee, cotton, a cucurbit,Douglas fir, dry bean eggplant, eucalyptus, fennel, flax, garden beans,garlic, gourd, grape, olive, okra, onion, leek, loblolly pine, melon,palm, lettuce, pea, peanut, pepper, potato, poplar, pine, pumpkin,radish, sunflower, safflower, sorghum, soybean, spinach, squash,strawberry, sugar beet, sweet gum, sweet potato, switch grass, tea,tobacco, tomato, triticale, turf grass, watermelon, ornamental, shrub,nut, chickpea, pigeon pea, millet, hops, and pasture grass plants.

“Embryo” is part of a seed, comprising precursor tissues (meristematictissues) for the leaves, stem, and root. Once the embryo begins to grow(germinate), it becomes a seedling plant.

“Meristem” or “meristematic tissue” comprises undifferentiated cells,the meristematic cells, which differentiate to produce multiple plantstructures including stem, roots, leaves, germline tissue and seeds“Explant” is a term used to refer to target material for transformationcomprising plant cells. The plant cells are defined as above.

The method of the present invention comprises contacting the plant cellswith an effective amount of polyethylene glycol (PEG) for a period oftime prior to transformation. Percent amounts of PEG described hereinare given by volume. In certain non-limiting embodiments, the PEG may bedissolved in sterile distilled water (SDW), Inoculation medium (INO, seeTable 7), or Bean Germination Medium (BGM, see Table 8). The effectiveamount of PEG of the present invention can vary depending on the cropand is generally in the range of about 1% to about 50%, including about1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50% and specificallyincluding all ranges derivable between any two such values. In certainnon-limiting embodiments, the average molecular weight of the PEG may befrom about 200 and about 35000, including about 200, 300, 400, 550, 600,1000, 1500, 2000, 3000, 3350, 4000, 6000, 8000, 10000, 20000, and 35000and specifically including all ranges derivable between any two suchvalues. In some embodiments, the molecular weight of PEG is 4000, 5000,6000, 7000, or 8000.

In some aspects of the present invention, the plant cells may becontacted with a composition comprising an effective amount of PEG, andone or more plant growth regulators (PGRs). Embryogenic culture responseis the product of interactions among genotype, developmental stage ofthe explant, culture conditions and growth regulator composition of theculture medium. During the process of culture initiation, an explantundergoes a process known as “de-differentiation”—a step in which theoriginal developmental stage of the explants is suitably modified withthe aid of growth regulators. Depending on the effectiveness of thegrowth regulators, this process may take hours or days, where deliveringthe optimum amount of growth regulator combination(s) is a key step. Onesignificant hurdle in this key step of delivering “growth regulators” isthat a very high dose of growth regulators may cause deleterious effectsto the explant. One aspect of the present invention provides a method tocontact explants with a high concentration of PGRs prior to culturing ona suitable culture medium without deleterious effects by combining thePGRs in a composition of PEG. This approach not only mitigatesdeleterious effect of high concentrations of growth regulators, but alsoenhances the de-differentiation process of plant cells.

Many PGRs are known in the art and could be used according to themethods of the present invention, such as auxins, including but notlimited to 4-CPA, 2,4-Dichlorophenoxyacetic acid (2,4-D), dichlorprop,fenoprop, indole-3-acetic acid (IAA), indole-3-butyric acid (IBA),naphthaleneacetamide, α-naphthaleneacetic acid, l-naphtholnaphthoxyacetic acid (NAA), potassium naphthenate, sodium naphthenate,and 2,4,5-trichlorophenoxy acetic acid, 3,6-dichloro-2-methoxybenzoicacid (dicamba), and 4-Amino-3,5,6-trichloropicolinic acid (picloram);antiauxins including but not limited to clofibric acid, and2,3,5-tri-iodobenzoic acid; cytokinins including but not limited to 2iP,benzyladenine, thidiazuron (TDZ), 6-benzylaminopurine (BAP), kinetin,and zeatin; defoliants including but not limited to calcium cyanamide,dimethipin, endothal, ethephon, merphos, metoxuron, pentachlorophenol,and tribufos; ethylene inhibitors including but not limited toaviglycine, and 1-methylcyclopropene; ethylene releasers including butnot limited to 1-aminocyclopropanecarboxylic acid, etacelasil, ethephon,and glyoxime; gibberellins including but not limited to gibberellic acid(GA3); herbicides including but not limited to glyphosate, glufosinate,DL-Phosphinothricin, 3,6-dichloro-2-methoxybenzoic acid, and2,4-Dichlorophenoxyacetic acid; growth inhibitors and retardantsincluding but not limited to abscisic acid, ancymidol, butralin,carbaryl, chlorphonium, chlorpropham, dikegulac, flumetralin,fluoridamid, fosamine, glyphosine, isopyrimol, jasmonic acid, maleichydrazide, mepiquat, piproctanyl, prohydrojasmon, propham,2,3,5-tri-iodobenzoic acid, chlormequat, daminozide, flurprimidol,mefluidide, paclobutrazol, tetcyclacis, and uniconazole; morphactinsincluding but not limited to chlorfluren, chlorflurenol,dichlorflurenol, and flurenol; growth stimulators including but notlimited to brassinolide, forchlorfenuron, and hymexazol; and/or otherunclassified plant growth regulators including but not limited tobenzofluor, buminafos, carvone, ciobutide, clofencet, cloxyfonac,cyanamide, cyclanilide, cycloheximide, cyprosulfamide, epocholeone,ethychlozate, ethylene, fenridazon, heptopargil, holosulf, inabenfide,karetazan, lead arsenate, methasulfocarb, prohexadione, pydanon,sintofen, triapenthenol, and trinexapac.

In certain embodiments of the present invention, the auxin is 2,4-D, andin other embodiments the auxin is picloram. In other embodiments thecytokinin is TDZ, and in still other embodiments the cytokinin is BAP.

One of skill in the art of plant cell culture and transformation wouldbe able to determine appropriate levels and/or ratios of plant growthregulators that are suitable for use for a specific plant species withthe present invention. For instance, levels of these or other PGRs witha functionally equivalent level of activity as, for instance, BAP and/or2,4-D in corn or in another plant species, may be determined by varyingthe levels of such growth regulators present in media to which explantsare contacted, and monitoring the growth of the explants and tissuesderived therefrom. Thus, if other PGRs are used, they would neverthelesscomprise a plant growth-regulatory effect equivalent to thesecontemplated amounts and ratios of the above listed PGRs.

In various non-limiting embodiments, the amount of PGRs used in thePEG-containing composition may range from 0.001 mg/L to about 30 mg/L,including about 0.01, 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30mg/L, and specifically including all ranges derivable between any twosuch values.

In certain non-limiting embodiments, immature corn embryo explants arecontacted with the PEG-containing composition for a period of about 1 to10 days, including about 2, 3, 4, 5, 6, 7, 8, 9, and 10 days, andspecifically including all ranges derivable between any two such values.In a specific embodiment, the explants are contacted with thecomposition for about 3 days.

In other embodiments, mature soy embryo explants comprising cells arecontacted with a PEG-containing composition for a period of about 30 to300 minutes, including about 30, 40, 50, 60, 70, 90, 100, 120, 180, 240,and 300 minutes, and specifically including all ranges derivable betweenany two such values. In a specific embodiment, the explants arecontacted with the composition for about 60 minutes.

In still other non-limiting embodiments, the explants are contacted withthe PEG-containing composition at a temperature from about 2 to 30degrees Celsius, including about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30degrees Celsius, and specifically including all ranges derivable betweenany two such values.

The ranges of amount, duration, temperature, and molecular weightdisclosed herein are non-limiting. Those of ordinary skill in the artcould carry out the present invention using an amount, duration,temperature or molecular weight below, above, or in between thosespecifically given, without departing from the scope and spirit of thepresent invention, depending upon the specific requirements of aparticular plant species.

The methods of present invention may further comprise a rinse step,wherein the plant cells which have been contacted with the polyethyleneglycol composition are rinsed with a non-PEG containing compositionprior to transformation. In various non-limiting aspects, the non-PEGcontaining composition may be water, or a medium such as ½ MS PL (Table1), ½ MS VI (Table 2), INO (Table 6), BGM (Table 8), or Agrobacteriumculture re-suspension, described elsewhere in the present disclosure. Incertain non-limiting embodiments, the rinse step may comprise one ormore rapid repetitious rinses with the desired rinse medium, or mayalternatively comprise a single rinse for a longer duration of 1 to 5minutes.

TABLE 1 ½ MS PL Medium Composition. Ingredient Source Amount per literMS Basal Salts Phytotech M524 2.165 g MS Vitamins Phytotech M533 103.1mg Glucose Phytotech G386 36 g Sucrose Phytotech S391 68.5 g ProlineFisher BP392-100 0.115 g pH to 5.4

TABLE 2 ½ MS VI Medium Composition. Ingredient Source Amount per literMS Basal Salts Phytotech M524 2.165 g MS Vitamins Phytotech M533 103.1mg Glucose Phytotech G386 10 g Sucrose Phytotech S391 20 g Proline SigmaP-5607 0.115 g pH to 5.4

In certain non-limiting aspects of the present invention, the plantcells may be immature embryos. When the immature embryos are of amonocot such as corn, the tissues to be substantially isolated areprovided in any suitable manner, for example attached to the ear or headon which the seeds mature. Monocot seeds may be removed from the ear orhead prior to substantially purifying the target tissue. Ears aretypically collected 10-12 days post pollination and surface sterilized.Methods for surface sterilization are well known in the art, andinclude, but are not limited to immersion in a solution containing aneffective concentration of ethanol, or sodium hypochlorite for aneffective period of time, for example from 1 to 15 minutes. Immatureembryos may be obtained by any suitable technique, including manually,or by mechanized or automated methods.

An opening in the pericarp or seed coat of the monocot seeds is providedto effectuate isolation of the desired explant. This may be accomplishedby any suitable technique, such as, but not limited to, making a hole,puncture, or incision with a needle, awl, blade, or other suitableimplement. In some applications of the method, no pericarp tissue needbe removed; in other applications, the opening of the pericarp mayinclude removal of at least part of the pericarp and possibly of somenon-embryo tissue (e.g., endosperm). Preferably, the opening issufficient to substantially separate the embryo from the seed, which maybe done in manually, by using a sterile tool such as a spatula to scoopout the endosperm and embryo, or by pushing on the side of the kernel,causing the embryo to emerge and be partially or completely exposed fromthe kernel. It may be necessary only to weaken the pericarp sufficiently(for example, by abrasion, or by other physical, chemical, or enzymatictreatment) so that application of force to the seed results insubstantial isolation of the target tissue, such as the embryo.

Methods of obtaining immature embryos by automated or mechanized meansare disclosed in U.S. Pat. No. 7,560,611. The method includes the stepof applying force to the seeds sufficient to substantially isolate thetarget tissue, such as an immature embryo, from the seeds, wherein thesubstantially isolated target tissue is suitable for genetictransformation and tissue culture. Force may be applied to multipleseeds consecutively or simultaneously. The applied force can becontinuous or non-continuous (for example, pulsed or wave-like force),and is generally mechanically applied, that is to say, the force isobtained through the use of a device or machine rather by human hand.The amount of force applied is preferably sufficient to overcome theadhesion of the target (e.g., embryo) and non-target (e.g., non-embryotissue such as endosperm) from each other, thus allowing separation ofthe target and non-target tissues. Any suitable force or forces may beemployed for removal of the target tissue from its seed, and multipleforces may be used in combination, sequentially or simultaneously.Suitable forces include, but are not limited to, fluid jet positivepressure, liquid jet positive pressure, mechanical positive pressure,negative pressure, aspiration, centrifugal force, linear acceleration,linear deceleration, fluid shear, fluid turbulent flow, and fluidlaminar flow. Fluid forces can be exerted by any fluid, gases or liquidsor combinations of both.

The method can further include the step of separating the substantiallyisolated target tissue, such as immature embryos, from associatednon-embryo tissue such as endosperm, glumes, and seed coat or pericarptissues. Methods for separating immature embryos from other tissues aredescribed in US Patent Application Publication No. 2009/0142837.Separation may be accomplished by one or more suitable techniques,including, but not limited to, separation by size exclusion (forexample, by filtration in one or more filtering steps), separation basedon hydrophobicity, hydrophilicity, lipophilicity, or other attractiveforces, and separation by mass or density differentials (for example,separation by centrifugation, settling, and decanting). The separationstep or steps can be optional, for example, where no additionalisolation of intact or partial embryos is necessary for their use intissue culture.

These methods to provide the substantially isolated target tissues, suchas corn embryos, that are suitable for genetic transformation or tissueculture can be automated, for example by employing robotic or mechanicalhandling of the corn ears or seeds, opening of the pericarp, applicationof force to the seed, or the optional separation steps. Such automationmay use optical or mechanical sensors to aid in positioning the cornears or seeds relative to the applied force or forces, or in theseparation steps. An apparatus for substantially isolating corn embryosis provided as disclosed in U.S. Pat. No. 7,560,611 that comprises atleast one aperture for guiding a fluid stream, wherein the fluid streamcontacts kernels on the corn ear and substantially isolates embryos fromthe kernels. Generally, it is preferred that the fluid stream contact asmany of the kernels in a given period of time as is convenient, so as tomore rapidly isolate embryos. The at least one aperture can include asingle aperture or multiple apertures (for example, single or multiplenozzles, which can include flat, round, oval, fan-shaped or otherpatterned nozzles, and adjustable, moving, or stationary nozzles), andcan generate a fluid flow of any suitable type and medium. Fluids may begases (such as air, nitrogen, or gas mixtures), liquids (such as water,physiological saline, or various culture media), or combinations.Suitable fluid flows include, but are not limited to, fluid jets (suchas single or multiple columnar jets; flat, cone-shaped, or fan-shapedjets or sprays; and sheet-like jets), laminar fluid flow, and turbulentfluid flow. Suitable fluid flows can result in one or more forces toremove the embryo from its kernel, including positive pressure ornegative pressure or both. The one or more forces may be applied tomultiple seeds consecutively or simultaneously, in a continuous ornon-continuous manner, and is generally applied mechanically and notmanually. Other suitable forces may be centrifugal force, linearacceleration, linear deceleration, and fluid shear. Such forces can beuniform or non-uniform, continuous or non-continuous (such as a pulsedor wave-like force) or in any combination thereof.

The apparatus may further include a means for moving the target tissuebeing substantially purified and the fluid stream, relative to eachother. For example, either the ear of corn containing seeds or the fluidstream, or both, may be moved. Various embodiments of the apparatus canbe used with single or multiple, intact or partial ears of corn. Forexample, the corn ear or ears can be secured to a holder or grasper,which is moved relative to the fluid stream. In other embodiments,however, the corn ear or ears need not be individually secured to aholder but can be freely movable so as to allow multiple kernels to becontacted by the force used to remove the embryos from the kernels. Themeans for moving at least one corn ear relative to the fluid stream canrotate the at least one corn ear and the at least one aperture relativeto each other, or can move the fluid stream along the longitudinal axisof the at least one corn ear, or can provide any suitablethree-dimensional movement of the at least one corn ear and the at leastone aperture relative to each other, such as a combination of rotationand longitudinal motion.

The apparatus can further include at least one separator for separatingtarget tissues from non-target tissues. For example, embryos may beseparated from non-embryo tissues, wherein the separated embryoscomprise at least some corn embryos suitable for genetic transformationor tissue culture. Separators can work by any suitable mechanism,including, but not limited to, separation by size exclusion (forexample, using a mesh, screen, perforated surface, or other devicecapable of excluding objects of a certain size), separation based onhydrophobicity or other attractive forces (for example, using amaterial, solid or fluid, that can attract or repel the embryos), andseparation by mass or density differentials (for example, using acentrifuge, or using solutions for differential settling). The separatorcan be optional, for example, where no additional isolation of intact orpartial embryos is necessary for their use in genetic transformation ortissue culture.

The substantially isolated (and optionally separated) immature embryosinclude at least some embryos, such as immature intact or partialembryos, suitable for tissue culture applications, transformation,callus formation, direct embryogenesis, formation of differentiatedplant tissue, formation of at least one mature plant, formation of atleast one fertile mature plant, and combinations of these processes, asdescribed above. The substantially isolated immature embryos andnon-embryo tissues may also be used for other purposes, such as, but notlimited to, genetic or biochemical analysis.

Combination apparatuses can optionally include a means for moving the atleast one corn ear relative to the source or sources of force (that isto say, the solid surface for applying mechanical positive pressure, theaperture for guiding a fluid flow, or the aperture for applying negativefluid pressure). Preferably the ear or ears is moved relative to thesource of force so that the force or forces contact as many of thekernels in a given period of time as is convenient, so as to morerapidly isolate embryos.

Combination apparatuses can further include at least one means forfurther separation of the substantially isolated immature embryossuitable for genetic transformation or tissue culture, wherein theseparated embryos comprise at least some corn embryos suitable forgenetic transformation or tissue culture. Separators can work by anysuitable mechanism, including, but not limited to, separation by sizeexclusion, separation based on attractive forces, and separation by massor density differentials.

In another aspect of the present invention, the plant cells are matureembryos. Mature embryo explants can be obtained from dry seed, forexample as disclosed in U.S. Patent Application Publication No.2008/0280361. These explants are referred to as dry-excised explants(DEEs). Dried wet explants may also be used, which are explants thathave been excised from seed following hydration/imbibition, and aresubsequently dehydrated and stored. Both of these types of explants havebeen shown to be fairly stable upon storage, but a rapid hydration stepprior to transformation may reduce their competency forbacterial-mediated transformation. Although not intending to be bound bya particular mechanism of action, the methods of the present inventionare useful in regulating the rate of hydration of embryonic explantsderived from dry seed, or explants that are dehydrated followingexcision of hydrated, or imbibed seeds, thereby improving the competencyof plant cells for bacterial-mediated transformation.

In various embodiments, explants comprising plant cells may be obtainedby either manual or mechanical methods. Prior to embryo excision, seedsmay be subjected to a sterilization step as well as a culling step, toavoid microbial contamination, to remove seeds with a high degree ofbacterial or fungal contamination, and also to remove seeds that may forany reason be unlikely to produce viable explant tissue for use with thepresent invention. Culling may be carried out, for example, based onparameters such as the size, color, or density of the seed or othercharacteristics, including chemical composition characteristics.Examples of culling methods may include the use of an automatic scaleafter size sorting. An optical sorter suitable for this purpose is theSortex 3000 Series Color Sorter (Buhler-Sortex KK, Yokohama, Japan).Other culling techniques may also be employed including culling bymoisture content.

In specific embodiments, excision is mechanically performed usingrollers that crush seeds applied to their faces, which can becounter-rotating. The gap between the rollers may be adjusted based onthe size of the applied seeds. Roller material may, for instance, beelastomeric or metallic. In certain embodiments, stainless steel rollershave been found to retain beneficial working qualities even followingrepeated and sustained use. In one embodiment, an explant may have aninternal moisture of about 3-25%, including about 3, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 16, 17, 18, 19, 20, 21, 22, 23, 24, and 25% internalmoisture, and specifically including all ranges derivable between anytwo such values. Seeds from which explants are to be prepared may beharvested at a predetermined internal moisture suitable for isolatingtransformable plant cells therefrom. Brittleness of seeds may be alteredby manipulating moisture content, allowing for efficient splitting ofseeds and preparation of explants. For instance, an internal moisturecontent such as 3% to 7% may be advantageous. Seeds may be held at suchmoisture contents or any other moisture content yielding stable storageconditions (and transformable explants) prior to use. Brittleness ofseeds may also be altered by exposing the seeds to low temperatures, forexample −20° C., or −80° C., or even colder, such as when contacted withliquid nitrogen (about −196° C.).

Dry explants of various ages may be used, including when explants arerelatively “young” in that they have been removed from seeds for lessthan a day, for example, from about 1 to 24 hours, such as about 2, 3,5, 7, 10, 12, 15, 20, or 23 hours prior to use. Explants may also bestored for longer periods, including days, weeks, months or even years,depending upon storage conditions used to maintain explant viability.Those of skill in the art in particular will understand that storagetimes may be optimized such that the quality and/or yield oftransformants as well as the efficiency of the transformation process ismaximized.

A dry seed or an explant may be first primed, for example, by imbibitionof a liquid such as water or a sterilization liquid, before beingredried, and later used for transformation and regeneration. The seed orthe explant may also be primed by raising the internal seed moisturecontent to greater than 30%, holding the seed or the explant at a timepoint, and then re-initiating imbibition at a later time point.Alternatively, the seed or the explant may be primed by raising theinternal moisture content to greater than 30%, storing the seed or theexplant for a predetermined period, drying the seed or the explant tothe internal moisture content of below 20%, and then re-initiatingimbibition.

Regenerable transformable explants may be harvested that contain no,some, or a part of each cotyledon remaining attached to the embryonictissue, for example as much as ¼ of the cotyledon. These explants areconsidered substantially similar, as they may each result in a stabletransformed plant. The explant should however contain at least some ofthe meristematic region of the embryo such that typically the explantcan produce a shoot within 12 weeks of the onset of tissue culturegrowth conditions.

A number of parameters for obtaining and handling explants may bevaried. Sterilization may be performed by contacting a seed or explantwith a liquid sterilizing agent. A seed or an explant may also becontacted with a gaseous sterilizing agent, or with an irradiatingsterilizing agent such as UV light. Alternatively, a seed or an explantmay be sterilized by subjecting the seed or the explant to a briefperiod of high temperatures so as to reduce the vigor of biologicalcontaminants such as adventitious bacteria and fungi on the surface ofthe seed or the explant without reducing the vigor of the seed or theexplant. This can be achieved at a temperature higher than 40° C., forexample from about 40° C. to about 90° C. The temperature can be raised,for instance, by either forced heated air or steam. Such temperaturescan be provided by dryers produced by Bry-Air Inc. (Sunbury, Ohio, USA).The addition of nystatin (50 ppm) and thiabendazole (10 ppm) dissolvedin DMSO (1.0 ml of DMSO per liter of INO) to a co-culture media (likeINO) may improve the health of explants, likely by controlling yeastsand fungi commonly found in and on seeds and can be a useful tool whenperforming large and/or automated tissue culture.

Moisture content of the seed at the time of excision may be varied, aswell as the temperature of the seed at the time of excision. Inaddition, a storage parameter following excision may be varied. Forinstance, the relative humidity under which explant storage occurs maybe varied. The explant storage temperature may also be varied, as wellas the duration of explant storage, and the composition of the medium inwhich the explant is stored. Further parameters that may be manipulatedinclude hydration and rehydration media compositions, incubationtemperature, length of time, and transformation methods, among others.

Following excision, methods and apparatuses for screening transformableexplant material from non-transformable damaged explants, cotyledons,seed coats, and other debris are employed as described in U.S. PatentApplication Publication No. 2008/0280361. The methods may be performedmanually, or may be partially or fully mechanized. For instance, one ormore steps of sieving may be performed, using sieves of appropriate sizebased on size of the seeds being crushed and the explants beingisolated. Bulk yield of crushed seed that has passed through the rollersmay be put through a series of separation sieves, such that unwantedlarge and small debris are separated from the desired explant by sizeexclusion. This may be effectively accomplished, for instance withsoybean material, using U.S. Standard sieves such as: #8 (2.36 mmopening), #10 (2.0 mm opening), #16 (1.18 mm opening), and others asappropriate (e.g. elongated window sieves such as 1/16″×¾″, 1/18″×¾″,1/19″×½″, or 1/20″×½″). Sieves with other opening sizes may befabricated as needed for given seed sizes, based on the size of materialbeing applied. The length of time for the screening process and thevigor of sieving may also be adjusted to enhance the throughput and/oryield of the process.

Other screening methods may also be utilized, such as by measuringdifferential buoyancy in solutions of explant material versus debris. Afraction of material that floats in an aqueous solution has been foundto be enriched for intact transformable explants.

The explant may be recovered from a hydrated seed, from dry storableseed, from a partial rehydration of dried hydrated explant, wherein“hydration” and “rehydration” is defined as a measurable change ininternal seed moisture percentage, or from a seed that is “primed”; thatis, a seed that has initiated germination but has been appropriatelyplaced in stasis pending favorable conditions to complete thegermination process. Those of skill in the art will be able to usevarious hydration methods and optimize length of incubation time priorto transformation. The resulting explant is storable and can germinateand or be transformed when appropriate conditions are provided. Thus thenew dry, storable meristem explant may be referred to as an artificialseed.

Following excision, one of skill in the art may store the explantaccording to the disclosed methods prior to subsequent use. Methods andparameters for drying, storing, and germinating seed are known in theart. Storage of excised explants comprising plant cells may be carriedout using modifications of such storage conditions as desired, includingtemperatures, for example, of from about −80° C. to about 60° C.Temperatures of about −20° C. to room temperature in particular havebeen found to function well.

After isolated embryo explants comprising cells are contacted with thecompositions of the present invention, the explants may be transformedby a selected heterologous DNA sequence, and transgenic plants may beregenerated therefrom. Transgenic plants may also be referred to astransgenic events. Various methods have been developed for transferringgenes into plant tissue including high velocity microprojection,microinjection, electroporation, direct DNA uptake, andbacterially-mediated transformation. Methods of using various bacteriaof species Rhizobiaceae as a vector for genetic transformation of plantsand/or plant cells, and regenerating transgenic plants therefrom areknown in the art. The host bacterial strain is often Agrobacteriumtumefaciens ABI, C58, LBA4404, EHA101, or EHA105 carrying a plasmidhaving a transfer function for the expression unit. In certainnon-limiting aspects of the present invention, Agrobacterium-mediatedtransformation and regeneration of a transgenic plant may be conductedas described in U.S. Pat. Nos. 5,824,877; 5,591,616; 5,981,840; and6,384,301 Other bacterial strains also known to those skilled in the artof plant transformation are contemplated for use in the presentinvention, for example Rhizobium-mediated transformation as described inU.S. Patent Application Publication 2007/0271627, Sinorhizobium sp.,Mesorhizobium sp., and Bradyrhizobium sp. (e.g. Broothaerts et al.,2005).

Means for preparing plasmids or vectors containing the desired geneticcomponents are well known in the art. Generally, the heterologous DNAsequence is combined with one or more genetic components to prepare anexpression unit. Often these expression units are provided with at leastone T-DNA border for transfer of the sequence to the plant cells. One ormore expression units are then inserted in a plasmid or vector which isthen mobilized into bacteria which are then contacted with the cells.The bacteria transfer the expression unit to the cell and the expressionunit get incorporated into the genome of the cell and then inheritedfrom one generation to the other.

The genetic components are incorporated into a plasmid or vectormolecule comprising at least one or more of the following geneticcomponents: (a) a promoter that functions in plant cells to cause theproduction of an RNA sequence, (b) a structural DNA sequence that causesthe production of an RNA sequence that encodes a product of commercialutility and/or a DNA sequence that causes the production of an RNAimolecule for inhibiting expression of a gene; and (c) a 3′non-translated DNA sequence that functions in plant cells to cause theaddition of polyadenylated nucleotides to the 3′ end of the RNAsequence.

Transcription of DNA into mRNA is regulated by a region of DNA usuallyreferred to as the “promoter”. The promoter region contains a sequenceof bases that signals RNA polymerase to associate with the DNA and toinitiate the transcription into mRNA using one of the DNA strands as atemplate to make a corresponding complementary strand of RNA. A numberof promoters that are active in plant cells have been described in theliterature. Such promoters would include but are not limited to thenopaline synthase (NOS) and octopine synthase (OCS) promoters that arecarried on Ti plasmids of Agrobacterium tumefaciens, the caulimoviruspromoters such as the cauliflower mosaic virus (CaMV) 19S and 35Spromoters and the Figwort mosaic virus (FMV) 35S promoter, and theenhanced CaMV35S promoter (e35S). A variety of other plant genepromoters that are regulated in response to environmental, hormonal,chemical, and/or developmental signals, also can be used for expressionof heterologous genes in plant cells, including, for instance, promotersregulated by (1) heat (Callis et al., 1988, (2) light (e.g., pea RbcS-3Apromoter, Kuhlemeier et al., (1989); maize RbcS promoter, Schaffner etal., (1991); (3) hormones, such as abscisic acid (Marcotte et al., 1989,(4) wounding (e.g., Wuni, Siebertz et al., 1989); or other signals orchemicals. Tissue specific expression is also known. As described below,it is preferred that the particular promoter selected should be capableof causing sufficient expression to result in the production of aneffective amount of the gene product of interest. Examples describingsuch promoters include without limitation U.S. Pat. No. 6,437,217 (maizeRS81 promoter), U.S. Pat. No. 5,641,876 (rice actin promoter), U.S. Pat.No. 6,426,446 (maize RS324 promoter), U.S. Pat. No. 6,429,362 (maizePR-1 promoter), U.S. Pat. No. 6,232,526 (maize A3 promoter), U.S. Pat.No. 6,177,611 (constitutive maize promoters), U.S. Pat. Nos. 5,322,938,5,352,605, 5,359,142 and 5,530,196 (35S promoter), U.S. Pat. No.6,433,252 (maize L3 oleosin promoter), U.S. Pat. No. 6,429,357 (riceactin 2 promoter as well as a rice actin 2 intron), U.S. Pat. No.5,837,848 (root specific promoter), U.S. Pat. No. 6,294,714 (lightinducible promoters), U.S. Pat. No. 6,140,078 (salt induciblepromoters), U.S. Pat. No. 6,252,138 (pathogen inducible promoters), U.S.Pat. No. 6,175,060 (phosphorus deficiency inducible promoters), U.S.Pat. No. 6,635,806 (gamma-coixin promoter), and U.S. patent applicationSer. No. 09/757,089 (maize chloroplast aldolase promoter). Additionalpromoters that may find use are a nopaline synthase (NOS) promoter(Ebert et al., 1987), the octopine synthase (OCS) promoter (which iscarried on tumor-inducing plasmids of Agrobacterium tumefaciens), thecaulimovirus promoters such as the cauliflower mosaic virus (CaMV) 19Spromoter (Lawton et al., 1987), the CaMV 35S promoter (Odell et al.,1985), the figwort mosaic virus 35S-promoter (Walker et al., 1987; U.S.Pat. Nos. 6,051,753; 5,378,619), the sucrose synthase promoter (Yang etal., 1990), the R gene complex promoter (Chandler et al., 1989), and thechlorophyll a/b binding protein gene promoter, PC1SV (U.S. Pat. No.5,850,019), and AGRtu.nos (GenBank Accession V00087; Depicker et al,1982; Bevan et al., 1983) promoters.

Promoter hybrids can also be constructed to enhance transcriptionalactivity (U.S. Pat. No. 5,106,739), or to combine desiredtranscriptional activity, inducibility and tissue specificity ordevelopmental specificity. Promoters that function in plants include butare not limited to promoters that are inducible, viral, synthetic,constitutive as described, and temporally regulated, spatiallyregulated, and spatio-temporally regulated. Other promoters that aretissue-enhanced, tissue-specific, or developmentally regulated are alsoknown in the art and envisioned to have utility in the practice of thisinvention.

The promoters used in the DNA constructs (i.e. chimeric/recombinantplant genes) of the present invention may be modified, if desired, toaffect their control characteristics. Promoters can be derived by meansof ligation with operator regions, random or controlled mutagenesis,etc. Furthermore, the promoters may be altered to contain multiple“enhancer sequences” to assist in elevating gene expression.

The mRNA produced by a DNA construct of the present invention may alsocontain a 5′ non-translated leader sequence. This sequence can bederived from the promoter selected to express the gene and can bespecifically modified so as to increase or decrease translation of themRNA. The 5′ non-translated regions can also be obtained from viralRNAs, from suitable eukaryotic genes, or from a synthetic gene sequence.Such “enhancer” sequences may be desirable to increase or alter thetranslational efficiency of the resultant mRNA. The present invention isnot limited to constructs wherein the non-translated region is derivedfrom both the 5′ non-translated sequence that accompanies the promotersequence. Rather, the non-translated leader sequence can be derived fromunrelated promoters or genes (see, for example U.S. Pat. No. 5,362,865).Examples of non-translation leader sequences include maize and petuniaheat shock protein leaders (U.S. Pat. No. 5,362,865), plant virus coatprotein leaders, plant rubisco leaders, GmHsp (U.S. Pat. No. 5,659,122),PhDnaK (U.S. Pat. No. 5,362,865), AtAnt1, TEV (Carrington and Freed,1990), and AGRtu.nos (GenBank Accession V00087; Bevan et al., 1983).Other genetic components that serve to enhance expression or affecttranscription or translational of a gene are also envisioned as geneticcomponents.

The 3′ non-translated region of the chimeric constructs may contain atranscriptional terminator, or an element having equivalent function,and a polyadenylation signal that functions in plants to cause theaddition of polyadenylated nucleotides to the 3′ end of the RNA. The DNAsequences are referred to herein as transcription-termination regions.The regions are required for efficient polyadenylation of transcribedmessenger RNA (mRNA). RNA polymerase transcribes a coding DNA sequencethrough a site where polyadenylation occurs. Examples of suitable 3′regions are (1) the 3′ transcribed, non-translated regions containingthe polyadenylation signal of Agrobacterium tumor-inducing (Ti) plasmidgenes, such as the nopaline synthase (NOS; Fraley et al., 1983) gene,and (2) plant genes such as the soybean storage protein genes and thesmall subunit of the ribulose-1,5-bisphosphate carboxylase (ssRUBISCO)gene. An example of a preferred 3′ region is that from the ssRUBISCO E9gene from pea (European Patent Application 0385 962).

The present invention can be used with any suitable plant transformationplasmid or vector containing a scorable, selectable, or screenablemarker and associated regulatory elements as described, along with oneor more nucleic acids expressed in a manner sufficient to confer aparticular desirable trait. Exemplary markers are known, and include butare not limited to GUS, green fluorescent protein (GFP), and luciferase(LUX), among others. Selectable or screenable markers function inregenerable plant tissue to produce a compound that confers upon theplant tissue resistance to an otherwise toxic compound. In certainembodiments, the vector comprises an aadA gene with associatedregulatory elements encoding resistance to spectinomycin in plant cells.In a particular embodiment, the aadA gene comprises a chloroplasttransit peptide (CTP) sequence that directs the transport of the aadAgene product to the chloroplast of a transformed plant cell. In otherembodiments, the vector comprises a spectinomycin resistance gene withappropriate regulatory elements designed for expression in a bacterialcell, such as an Agrobacterium cell, so that the selection reagent maybe added to a co-cultivation medium, and allowing obtention oftransgenic plants for instance without further use of the selectiveagent after the co-culture period. Examples of suitable genes conferringtraits of agronomic interest envisioned by the present invention wouldinclude but are not limited to genes for disease, insect, or pesttolerance, herbicide tolerance, genes ior quality improvements such asyield, nutritional enhancements, environmental or stress tolerances, orany desirable changes in plant physiology, growth, development,morphology or plant product(s) including starch production (U.S. Pat.Nos. 6,538,181; 6,538,179; 6,538,178; 5,750,876; 6,476,295), modifiedoils production (U.S. Pat. Nos. 6,444,876; 6,426,447; 6,380,462), highoil production (U.S. Pat. Nos. 6,495,739; 5,608,149; 6,483,008;6,476,295), modified fatty acid content (U.S. Pat. Nos. 6,828,475;6,822,141; 6,770,465; 6,706,950; 6,660,849; 6,596,538; 6,589,767;6,537,750; 6,489,461; 6,459,018), high protein production (U.S. Pat. No.6,380,466), fruit ripening (U.S. Pat. No. 5,512,466), enhanced animaland human nutrition (U.S. Pat. Nos. 6,723,837; 6,653,530; 6,5412,59;5,985,605; 6,171,640), biopolymers (U.S. Pat. Nos. RE37,543; 6,228,623;5,958,745 and U.S. Patent Publication No. US20030028917). Alsoenvironmental stress resistance (U.S. Pat. No. 6,072,103),pharmaceutical peptides and secretable peptides (U.S. Pat. Nos.6,812,379; 6,774,283; 6,140,075; 6,080,560), improved processing traits(U.S. Pat. No. 6,476,295), improved digestibility (U.S. Pat. No.6,531,648) low raffinose (U.S. Pat. No. 6,166,292), industrial enzymeproduction (U.S. Pat. No. 5,543,576), improved flavor (U.S. Pat. No.6,011,199), nitrogen fixation (U.S. Pat. No. 5,229,114), hybrid seedproduction (U.S. Pat. No. 5,689,041), fiber production (U.S. Pat. Nos.6,576,818; 6,271,443; 5,981,834; 5,869,720) and biofuel production (U.S.Pat. No. 5,998,700). Any of these or other genetic elements, methods,and transgenes may be used with the invention as will be appreciated bythose of skill in the art in view of the instant disclosure.

Alternatively, the DNA sequences of interest can affect these phenotypesby encoding a an RNA molecule that causes the targeted inhibition ofexpression of an endogenous gene via gene silencing technologies such asantisense-, co-suppression-mediated mechanisms, RNAi technologiesincluding miRNA (e.g., U.S. Patent Application Publication2006/0200878).

Exemplary nucleic acids that may be introduced by the methodsencompassed by the present invention include, for example, DNA sequencesor genes from another species, or even genes or sequences that originatewith or are present in the same species, but are incorporated intorecipient cells by genetic engineering methods rather than classicalreproduction or breeding techniques. However, the term “exogenous” isalso intended to refer to genes that are not normally present in thecell being transformed, or perhaps simply not present in the form,structure, etc., as found in the transforming DNA segment or gene, orgenes that are normally present yet that one desires, e.g., to haveover-expressed. Thus, the term “exogenous” gene or DNA is intended torefer to any gene or DNA segment that is introduced into a recipientcell, regardless of whether a similar gene may already be present insuch a cell. The type of DNA included in the exogenous DNA can includeDNA that is already present in the plant cell, DNA from another plant,DNA from a different organism, or a DNA generated externally, such as aDNA sequence containing an antisense message of a gene, or a DNAsequence encoding a synthetic or modified version of a gene.

The T-DNAs may be bound by RB and/or LB sequences or may have no bordersequences. The sequences that may be transferred into a plant cell maybe present on one transformation vector in a bacterial strain beingutilized for transformation. In another embodiment, the sequences may bepresent on separate transformation vectors in the bacterial strain. Inyet another embodiment, the sequences may be found in separate bacterialcells or strains used together for transformation.

The DNA constructs used for transformation in the methods of presentinvention may also contain the plasmid backbone DNA segments thatprovide replication function and antibiotic selection in bacterialcells, for example, an Escherichia coli origin of replication such asori322, a broad host range origin of replication such as oriV or oriRi,and a coding region for a selectable marker such as Spec/Strp thatencodes for aminoglycoside adenyltransferase (aadA) conferringresistance to spectinomycin or streptomycin (e.g. U.S. Pat. No.5,217,902. In light of this disclosure, numerous other possibleregulatory elements, and other sequences of interest will be apparent tothose of skill in the art. Therefore, the foregoing discussion isintended to be exemplary rather than exhaustive.

Those of skill in the art are aware of the typical steps in the plantAgrobacterium-mediated transformation process. The Agrobacterium can beprepared either by inoculating a liquid such as Luria Burtani (LB) mediadirectly from a glycerol stock or streaking the Agrobacterium onto asolidified media from a glycerol stock, allowing the bacteria to growunder the appropriate selective conditions, generally from about 26°C.-30° C., or about 28° C., and taking a single colony or a small loopof Agrobacterium from the plate and inoculating a liquid culture mediumcontaining the selective agents. Those of skill in the art are familiarwith procedures for growth and suitable culture conditions forAgrobacterium as well as subsequent inoculation procedures. The densityof the Agrobacterium culture used for inoculation and the ratio ofAgrobacterium cells to explant can vary from one system to the next, andtherefore optimization of these parameters for any transformation methodis expected.

Typically, an Agrobacterium culture is inoculated from a streaked plateor glycerol stock and is grown overnight and the bacterial cells arewashed and resuspended in a culture medium suitable for inoculation ofthe explant. Suitable inoculation media for the present inventioninclude, but are not limited to ½ MMS PL or ½ MS VI for immature embryoexplants (see Tables 1 and 2, respectively, and INO (see Table 6) formature embryo explants.

The next stage of the Agrobacterium mediated transformation process isthe inoculation. In this stage the explants and Agrobacterium cellsuspensions are mixed together. In embodiments of the present inventionwhere the embryo explants are of immature corn embryos, embryo explantsare placed directly into the inoculation medium containing theAgrobacterium. Embryos are cultured in inoculation media for less than30 min. The inoculation is generally performed at a temperature of about15° C. to 30° C., or about 23° C. to 28° C. The inoculation can also bedone by isolating the immature embryos directly onto the co-culturemedium (described below) and then spotting 1 μL of Agrobacteriumsolution onto the embryo or alternatively placing a piece of filterpaper saturated in Agrobacterium solution over the top of the embryosfor about 5 to 60 minutes. The filter paper and any excess solution arethen removed before co-culture.

In certain embodiments, at the time, or subsequent to the time that aheterologous DNA is contacting the explant, the explant may be contactedby one or more plant growth regulators (PGRs). Many PGRs are known inthe art and are contemplated in such embodiments, such as auxins,antiauxins, cytokinins, gibberellins, herbicides, growth inhibitors andretardants, morphactins, growth stimulators and/or other unclassifiedplant growth regulators, as listed elsewhere in the present disclosure.

After inoculation, any excess Agrobacteriwnum suspension can be removedand the Agrobacterium and target plant material are co-cultured. Theco-culture refers to the time post-inoculation and prior to transfer toa delay or selection medium. Any number of plant tissue culture mediacan be used for the co-culture step. In certain embodiments, afterinoculation with Agrobacterium the plant tissues are cultured on asemi-solid MS-based, or reduced salt % MS-based semi-solid co-culturemedium with a gelling agent such as agarose, or a low EEO agarose (Table3).

TABLE 3 MS Based semi-solid co-culture medium. ½ MS Based co- MS Basedco-culture culture medium medium Ingredient Source Amount per liter MSBasal Salts Phytotech M524 2.165 g 4.33 g MS Vitamins Phytotech M533103.1 mg 103.1 mg Thiamine HCL Sigma T-3902 0.5 mg 0.5 mg 2,4-DPhytotech D295 3 mg 0.5 mg Glucose Phytotech G386 10 g 0 SucrosePhytotech S391 20 g 30 g Proline Sigma P-5607 0.115 g 1.38 g CasaminoAcids Difco DF0288-17 0 0.5 g Agarose, Low EEO Sigma A-6013 5.5 g 5.5 gAcetosyringone Aldrich, D134406 40 mg 40 mg Silver Nitrate Sigma S-65063.4 mg 3.4 mg Carbenicillin Phytotech C346 0 50 mg pH 5.2 5.8

The co-culture is typically performed for about one to three days or forless than 24 hours at a temperature of about 18° C. to 30° C., or about20° C. to 25° C. The co-culture can be performed in the light or inlight-limiting conditions. Usually, the co-culture is performed inlight-limiting conditions. “Light-limiting conditions” as used hereinare defined as any conditions that limit light during the co-cultureperiod including but not limited to covering a culture dish containingthe plant and Agrobacterium mixture with a cloth, foil, or placing theculture dishes in a black bag, or placing the cultured cells in a darkroom. Lighting conditions can be optimized for each plant system as isknown to those of skill in the art.

In embodiments of the present invention where the embryo explants are ofmature soy embryos, embryo explants are exposed to the preparedinoculum, and briefly exposed to sonication energy from a standardlaboratory water bath cleaning sonicator such as L&R Ultrasonics QS 140(L&R Manufacturing Co., Kearny, N.J.); or a Honda W113 sonicator (Honda,Denshi Japan) for 20 seconds. After the brief sonication step, explantsare drained of originating inoculum and transferred to fresh PLANTCONseach containing filter paper moistened with INO media, usually withinseveral hours after commencement of transfection. Explants are thenincubated in a lighted chamber (generally 16 hours of light at ≥5 uE) atapproximately 23 to 28 C for 1 to 5 days. Co-culture and subsequentsteps may be performed in dark conditions, or in the light, e.g. lightedPercival incubators, for instance for 2 to 5 days (e.g. a photoperiod of16 hours of light/8 hours of dark, with light intensity of ≥5 μE, suchas about 5-200 μE or other lighting conditions that allow for normalplastid development) at a temperature of approximately 23 to 25° C., andmay be performed at up to about 35° C. or 40° C.

After co-culture with Agrobacterium or after bombardment with themicroprojectile, the explants can be placed directly onto regenerationmedia, typically containing a selective agent. Explants (regardless oftransformation method) are placed on selective media for from about 7 toabout 42 days, or from about 7 to about 30 days, or from about 7 toabout 21 days, or from about 7 to about 14 days. A variety of tissueculture media and transfer requirements are known that can beimplemented and optimized to support plant tissue growth and developmentfor plant transformation and recovery of transgenic plants. These tissueculture media can either be purchased as a commercial preparation orcustom prepared and modified by those of skill in the art. Examples ofsuch media include, but are not limited to those described by Murashigeand Skoog, (1962); Chu et al., (1975); Linsmaier and Skoog, (1965);Uchimiya and Murashige, (1962); Gamborg et al., (1968); Duncan et al.,(1985); McCown and Lloyd, (1981); Nitsch and Nitsch (1969); and Schenkand Hildebrandt, (1972), or derivations of these media supplementedaccordingly. Those of skill in the art are aware that media and mediasupplements such as nutrients and growth regulators for use intransformation and regeneration are usually optimizable for a particulartarget crop or variety of interest. Tissue culture media may besupplemented with carbohydrates such as, but not limited to, glucose,sucrose, maltose, mannose, fructose, lactose, galactose, and/ordextrose, or ratios of carbohydrates. Reagents are commerciallyavailable and can be purchased from a number of suppliers (see, forexample Sigma Chemical Co., St. Louis, Mo.; and PhytoTechnologyLaboratories, Shawnee Mission, Kans.). Additional appropriate mediacomponents can be added to the selection or delay medium to inhibitAgrobacterium growth. Such media components can include, but are notlimited to, antibiotics such as carbenicillin or cefotaxime. Thecultures are subsequently transferred to a media suitable for therecovery of transformed plantlets. Those of skill in the art are alsoaware of the numerous modifications in selective regimes, media, andgrowth conditions that can be varied depending on the plant system andthe selective agent. Typical selective agents include but are notlimited to antibiotics such as geneticin (G418), kanamycin, paromomycin,spectinomycin, or other chemicals such as glyphosate or otherherbicides. In a specific embodiment of the present invention, theselective agent is spectinomycin. Spectinomycin resistant shoots thathave green buds or leaves are screenable or scoreable as beingspectinomycin resistant. They may be placed in soil or on a soilsubstitute such as on a rooting medium, in the presence or absence ofthe selective agent. Shoots elongating from such an explant areroutinely shown to be transgenic and give rise to R_(r) and subsequentprogeny that are transgenic, while the roots developing from suchexplants may be transgenic or non-transgenic. Thus, a plant comprising atransgenic shoot and a partly or fully non-transgenic root system isalso contemplated. Alternatively, a method for regenerating a wholeplant from transgenic shoots from transformed meristematic tissue whileroots are non-transgenic, by culturing of transformed tissue on a mediumlacking a selective agent, is also contemplated, such as disclosed inU.S. Patent Application Publication No. 2008/0057512.

These methods can also be done in one or more containers and the processmay be manual, or automated using state of the art automation. Media andculture conditions disclosed in the present invention can be modified orsubstituted with nutritionally equivalent components, or similarprocesses for selection and recovery of transgenic events, and stillfall within the scope of the present invention. A transgenic plantformed using Agrobacterium transformation methods typically (althoughnot always) contains a single simple recombinant DNA sequence (singlecopy) inserted into one chromosome and is referred to as a transgenicevent. Such transgenic plants can be referred to as being heterozygousfor the inserted exogenous sequence. A transgenic plant homozygous withrespect to a transgene can be obtained by sexually mating (selfing) anindependent segregant transgenic plant that contains a single exogenousgene sequence to itself, for example an R₀ plant, to produce R₁ seed.One fourth of the R₁ seed produced will be homozygous with respect tothe transgene. Germinating R₁ seed results in plants that can be testedfor zygosity, typically using a SNP assay or a thermal amplificationassay that allows for the distinction between heterozygotes andhomozygotes (i.e., a zygosity assay).

To confirm the presence of the exogenous DNA or “transgene(s)” in thetransgenic plants a variety of assays may be performed. Such assaysinclude, for example, “molecular biological” assays, such as Southernand northern blotting and PCR™, INVADER assays, Recombinase PolymeraseAmplification (RPA) method (see for example U.S. Pat. No. 7,485,428);“biochemical” assays, such as detecting the presence of a proteinproduct, e.g., by immunological means (ELISAs and western blots) or byenzymatic function; plant part assays, such as leaf or root assays; andalso, by analyzing the phenotype of the whole regenerated plant.

Once a transgene has been introduced into a plant, that gene can beintroduced into any plant sexually compatible with the first plant bycrossing, without the need for ever directly transforming the secondplant. Therefore, as used herein the term “progeny” denotes theoffspring of any generation of a parent plant prepared in accordancewith the instant invention, wherein the progeny comprises a selected DNAconstruct. A “transgenic plant” may thus be of any generation.“Crossing” a plant to provide a plant line having one or more addedtransgenes or alleles relative to a starting plant line is defined asthe techniques that result in a particular sequence being introducedinto a plant line by crossing a starting line with a donor plant linethat comprises a transgene or allele. To achieve this one could, forexample, perform the following steps: (a) plant seeds of the first(starting line) and second (donor plant line that comprises a desiredtransgene or allele) parent plants; (b) grow the seeds of the first andsecond parent plants into plants that bear flowers; (c) pollinate aflower from the first parent plant with pollen from the second parentplant; and (d) harvest seeds produced on the parent plant bearing thefertilized flower.

Another aspect of the present invention is a transgenic plant and anyplant parts thereof as defined elsewhere in the present disclosure,created using the methods of the present invention disclosed herein.

EXAMPLES

Those of skill in the art will appreciate the many advantages of themethods and compositions provided by the present invention. Thefollowing examples are included to demonstrate certain embodiments ofthe invention. Those of skill in the art should, in light of the presentdisclosure, appreciate that many changes can be made in the specificembodiments that are disclosed and still obtain a like or similar resultwithout departing from the spirit and scope of the invention. Allreferences cited herein are incorporated herein by reference to theextent that they supplement, explain, provide a background for, or teachmethodology, techniques, or compositions employed herein.

Example 1 Enhanced Transformation of Soybean Cells Contacted with PEG inWater

This example demonstrates enhanced transformation of dry soybean embryosthat were contacted with a composition containing PEG in steriledistilled water (SDW). Soybean cv. A3525 dry embryos were excisedaccording to the method described in U.S. Patent Application PublicationNo. 2008/0280361 and contacted for an hour with various amounts of 5,10, 20, or 50% PEG-4000 dissolved in SDW. The embryos were rinsed 5-6×with SDW and transformed according to the methods described in U.S.Patent Application Publication No. 2009/0138985. The embryos weretransformed with a 2T transformation vector having an OriRi or OriVreplication of origin and contained in ABI or AB30 strain ofAgrobacterium. The embryos were regenerated on spectinomycin selectionmedium. As shown in Table 4, embryos contacted with 5, 10, and 20% PEGcomposition generally showed enhanced transformation frequency, andenhanced frequency of single copy events, compared to embryos contactedwith INO medium or SDW alone. Embryos that were contacted with 50% PEGalso demonstrated an improved TF (7%) as compared to embryos that werecontacted with SDW (TF=2.3%). The 50% PEG experiment was done with anAB30/OriRi construct. See Table 5 for spectinomycin selection mediumcomposition for soybean.

TABLE 4 Transformation frequency of soybean cells contacted with PEG inwater compared to water or INO medium. Treatment TF % TF % (ABI/OriRi)(AB30/OriV) INO medium 5.9 6.2 H2O 2.3 1.7  1% PEG in H2O 1.9 3.7  5%PEG in H2O 8.1 5.2 10% PEG in H2O 14.3 12.3 20% PEG in H2O 19.6 12.2

TABLE 5 Spectinomycin selection medium composition for soybean. Amountper Ingredient Source liter LM Woody Plant Phytotech L449 2.41 g Mediumw/Vitamins Sucrose Phytotech S391 20 g Calcium Gluconate Sigma G-46251.29 g Agargel Sigma A-3301 4 g Carbenicillin (40 mg/mL stock) PhytotechC346 5 mL Timentin (100 mg/mL stock) Duchefa T0190 1 mL Cefotaxime (50mg/mL stock) Midwest NDC0039-0019-10 4 mL Spectinomycin (50 mg/mL stock)Sigma S-4014 3 mL

Example 2 Enhanced Transformation of Soybean Cells Contacted with PEG inINO Medium

This example demonstrates enhanced transformation of dry soybean embryosthat were contacted with a composition containing PEG in INO or INOmedium. Soybean cv. A3525 dry embryos were excised according to themethod described in U.S. Patent Application Publication No. 2008/0280361and contacted for an hour with 20% PEG-4000 dissolved in INO. Theembryos were rinsed 5-6× with plain INO and transformed according to themethods described in U.S. Patent Application Publication No.2009/0138985. The embryos were transformed with a 2T transformationvector having an OriRi replication of origin and contained in AB30strain of Agrobacterium. The embryos were regenerated on spectinomycinselection medium. As shown in Table 6, embryos contacted with 20% PEGcomposition generally showed enhanced transformation frequency comparedto embryos contacted with INO medium alone in two different experiments.See Table 7 for INO medium composition.

TABLE 6 Transformation frequency of soybean cells contacted with PEG inINO compared to INO. Treatment # Explants # Events to Soil TF % INO 4135210 5.1 20% PEG in INO 4049 317 7.8 INO 4149 163 3.9 20% PEG in INO 4110341 8.3

TABLE 7 Composition of inoculation medium (INO) Ingredient Amount perliter Magnesium sulfate (Fisher M63) 0.1 g Ammonium sulfate (FisherA702) 53.6 mg Sodium phosphate monohydrate 60 mg (Fisher S369-500)Calcium chloride (Sigma C-3881) 60 mg Boric acid (Fisher A73-3) 0.3 mgManganese sulfate (Sigma I-2550) 1 mg Zinc sulfate heptahydrate 0.2 mg(Sigma Z-1001) Potassium iodide (Sigma P-8166) 0.075 mg Sodium Molybdatedihydrate 0.025 mg (Sigma S-6646) Cupric sulfate (Fisher C493-500) 2.5μg Cobalt chloride hexahydrate 2.5 μg (Sigma C-2911) Sequestrene (Ciba964603) 2.8 mg Potassium nitrate (Sigma P-8291) 1 g Glucose (PhytotechG386) 30 g MES (Sigma M8250) 3.9 g Bring volume to 1 L with de-ionizeddistilled water pH with KOH to 5.4 Autoclave Add sterile vitamin stockcontaining the following Myo-inositol (Sigma I-3011) 10 mg Nicotinicacid (Sigma N-0765) 0.1 mg Pyridoxine HCl (Sigma P-8666) 0.1 mg ThiamineHCl (Sigma T-3902) 1 mg

Example 3 Enhanced Transformation of Soybean Cells Contacted with PEG inBGM

This example demonstrates enhanced transformation of dry soybean embryosthat were contacted with a composition containing PEG in BeanGermination Medium (BGM—Table 8) compared to BGM alone. Soybean cv.A3525 dry embryos were excised according to the method described in U.S.Patent Application Publication No. 2008/0280361 and contacted for anhour with 10% or 20% PEG-4000 dissolved in BGM. The embryos were rinsed5-6× with BGM and transformed according to the methods described in U.S.Patent Application Publication No. 2009/0138985. The embryos weretransformed with a 2T transformation vector having an OriV replicationof origin and contained in AB30 strain of Agrobacterium. The embryoswere regenerated on spectinomycin selection medium. As shown in Table 9,embryos contacted with 10% and 20% PEG composition showed enhancedtransformation frequency compared to embryos contacted with BGM alone.

TABLE 8 Composition of Bean Germination Medium (BGM). Ingredient SourceAmount per liter NH₄NO₃ (Sigma A-3795) 240 mg KNO₃ (Sigma P-8291) 505 mgCaCl₂ • 2H₂O (Sigma C3881) 176 mg MgSO₄ • 7H₂O (Fisher M63) 493 mgKH₂PO₄ (Fisher BP362-500) 27 mg H₃BO₃ (Fisher BP168-1) 1.86 mg Na₂MoO₄ •2H₂O (Sigma S-6646) 0.216 mg MnSO₄ • H₂O (Sigma I-2550) 5.07 mg ZnSO₄ •7H₂O (Sigma Z1001) 2.58 mg FeSO₄ • 7H₂O (Sigma F8263) 2.502 mg KI (SigmaP-8256) 0.249 mg Na₂EDTA • 2H₂O (Fisher BP120) 3.348 mg CuSO₄ • 5H₂O(Sigma C-3036) 0.0008 mg CoCl₂ • 6H₂O Sigma C-2911) 0.0008 mg ThiamineHCl Sigma C-2911) 1.34 mg Nicotinic Acid (Sigma N-0765) 0.5 mgPyridoxine HCl (Sigma P-8666) 0.82 mg Bravo (75% WP) (Carlin) 30 mgCaptan (50% WP) (Carlin 10-0250) 30 mg Sucrose (Phytotech S391) 25000 mgpH 5.8

TABLE 9 Transformation frequency of soybean cells contacted with PEG inBGM compared to BGM medium. Treatment Explants # R0 Plants TF BGM 163512 0.73% BGM 10% PEG 1562 39 2.50% BGM 20% PEG 1487 38 2.56%

Example 4 Effect of PEG on TF Compared to Other Osmotic Compounds

This example demonstrates the effect of other osmotic compounds on TFcompared to PEG. It also compares TF enhancement of treatments withvarious PEG species. Soybean cv. A3555 dry embryos were excisedaccording to the method described in U.S. Patent Application PublicationNo. 2008/0280361 and contacted for an hour with various amounts ofPEG-4000, PEG-6000, PEG-8000, Mannitol, Sorbitol, or Glycerol dissolvedin SDW. The embryos were then rinsed 5-6× with SDW and transformedaccording to the methods described in U.S. Patent ApplicationPublication No. 2009/0138985. The embryos were transformed with a 2Ttransformation vector having an OriRi replication of origin andcontained in AB30 strain of Agrobacterium. The embryos were regeneratedon spectinomycin selection medium. As shown in Table 10, embryoscontacted with 10% and 20% PEG compositions showed enhancedtransformation frequency compared to embryos treated with other osmoticcompounds, or with water alone.

TABLE 10 Transformation frequency soybean cells contacted with PEGcompared to other osmotic compounds. Treatment Initial Explants # R0Plants TF H2O 1562 76 4.90% 20% PEG4000 1165 177 15.20% 10% PEG6000 108899 9.10% 20% PEG6000 1280 220 17.20% 10% PEG8000 1485 113 7.60% 20%PEG8000 1478 215 14.50% 10% mannitol 1008 5 0.50% 10% sorbitol 1034 40.40% 20% sorbitol 906 3 0.30% 10% glycerol 174 4 2.30% 20% glycerol 2050 0.00%

Example 5 PEG Regulates the Rate of Hydration in Dry Embryo Explants

This example demonstrates that PEG is useful for regulating the rate ofhydration of dry embryo explants. Soybean cv. A3525 dry embryos wereexcised according to the method described in U.S. Patent ApplicationPublication No. 2008/0280361. The explants were then contacted witheither 20% PEG4000 dissolved in INO, or INO alone. The rate of moistureintake was measured by taking samples of the explants at timed intervalsduring treatment and then subjecting them to a destructivegravimetric-based oven test in which explants were dried in a ˜100° C.oven for approximately two days. Percent moisture was determined fromtheir weight loss. A first study examined hydration rates at 1, 4, and24 hours, while a second study examined rate of hydration rates at 0,15, 30, 45, and 60 minutes, as shown in tables 11 and 12, respectively.Both studies indicate the rate of moisture intake by dry soy embryosexplants that are contacted with 20% PEG4000 is reduced. Regulatinghydration with

TABLE 11 Hydration of dry embryo explants contacted with PEG compared toINO medium recorded in hourly intervals. Time Moisture Content (hours)INO 20% PEC4000 in INO  0  5.8% +/− 0.1%  5.8% +/− 0.1%  1 64.4% +/−0.3% 55.2% +/− 0.2%  4 63.6% +/− 0.4% 58.7% +/− 0.3% 24 64.6% 57.7%

TABLE 12 Hydration of dry embryo explants contacted with PEG compared toINO medium recorded in 15 minute intervals. Time Moisture Content(minutes) INO 20% PEG4000 in INO  0  5.8% +/− 0.1%  5.8% +/− 0.1% 1551.2% +/− 0.5% 44.6% +/− 0.5% 30 59.6% +/− 0.6% 52.1% +/− 0.4% 45 60.7%+/− 2.0% 54.5% +/− 0.9% 60 63.20% 58.10%

Example 6 PEG-Treatment Reduces Exudates Release of Embryo Explants

This example demonstrates that dry-excised embryo explants contactedwith PEG in water release less exudates compared to those contacted withwater alone. Soybean cv. A3555 dry embryos were excised according to themethod described in U.S. Patent Application Publication No. 2008/0280361and contacted for an hour with 20% PEG-4000 in SDW, or SDW alone. Theexplants, in their respective media were placed on an orbital shaker, at75 RPM and incubated at room temperature. Samples of the media from eachgroup were taken after 0, 15, 30, 45, and 60 minutes of incubation, andanalyzed for exudates as measured by optical density (absorbance at 300nm, blanked with appropriate medium) and conductivity (in microsiemens).As shown in Table 13, optical density of exudates was much higher inwater control samples compared to samples from the 20% PEG treatment.The same was true of conductivity. Note: Readings denoted with anasterisk (*) were done with 1/10 dilutions of samples and thenmultiplied by 10 because spectrophotometer used had a maximum range of3.295.

TABLE 13 Optical density and conductivity of explants treated with PEGcompared water treatment. Optical Density A300 nm Conductivity (μS) 20%PEG- 20% PEG-4000 Time (minutes) H20 4000 H20 4000 Media Blank 0 0 2.6124  0 0.512 0.203 68.5 148.5 15 3.74* 1.132 748.6 336.2 30 6.25* 1.7561011 447.8 45 7.06* 2.411 1152 519.8 60 7.46* 2.849 1320 557.4

Example 7 Effect of PEG Treatment on Transformation Frequency ofHydrated Soybean Embryos

This example demonstrates the effect of PEG treatment on transformationfrequency of hydrated soybean embryos when contacted prior toAgrobacterium-mediated transformation. Soybean cv. A3525 soybean embryoswere excised according to the method described in U.S. PatentApplication Publication No. 2008/0280361 and contacted for an hour withvarious amounts of PEG-4000 in SDW. The embryos were rinsed 5-6× withSDW and transformed according to the methods described in U.S. Pat. No.7,402,734. The embryos were transformed with a 2T transformation vectorhaving an OriRi replication of origin and contained in AB30 strain ofAgrobacterium. The embryos were regenerated on spectinomycin selectionmedium. As shown in Table 14, PEG treatment did not appear to be usefulin enhancing TF of already hydrated soybean mature embryos. It may bethat PEG treatment is beneficial to dry embryos, and immature embryos asexemplified with the corn immature embryos below.

TABLE 14 Transformation frequency of hydrated soybean cells contactedwith PEG in water compared to water. Treatment Explants # Rooted TF H2O394 66 16.75%  1% PEG 436 43 9.86%  5% PEG 393 45 11.45% 10% PEG 374 4211.23% 20% PEG 387 25 6.46%

Example 8 Effect of Use of PEG During Co-Culture on TransformationFrequency

This example demonstrates the effect of PEG composition duringco-culture on transformation frequency. Soybean cv. A3555 dry embryoswere excised according to the method described in U.S. PatentApplication Publication No. 2008/0280361 and contacted for an hour with20% PEG-4000 dissolved in SDW. The embryos were rinsed 5-6× with SDW andtransformed according to the methods described in U.S. PatentApplication Publication No. 2009/0138985. The embryos were transformedwith a 2T transformation vector having an OriRi replication of originand contained in AB30 strain of Agrobacterium. The transformed cellswere then co-cultured in INO medium containing 1, 5, 10, or 20% PEG4000. Co-culture medium also contained 1 ppm TDZ. The embryos wereregenerated on spectinomycin selection medium. As shown in Table 15,treatments in which PEG was included in co-culture medium yielded alower TF compared to treatments in which PEG was used prior toco-culture. This suggests that while PEG treatment prior totransformation improves competency of cells for bacterially-mediatedtransformation, PEG during co-culture appears to reduce TF.

TABLE 15 Transformation frequency of soybean cells contacted with PEGduring co-culture. Initial # R0 Treatment Co-culture Medium ExplantsPlants TF H2O INO 1562 76 4.90% 20% PEG4000 INO 1165 177 15.20% 20%PEG4000  1% PEG4000 in INO 922 84 9.10% 20% PEG4000  5% PEG4000 in INO782 52 6.60% 20% PEG4000 10% PEG4000 in INO 922 17 1.80% 20% PEG4000 20%PEG4000 in INO 762 0 0.00%

Example 9 Enhanced Callus Production of Corn Embryos Contacted with PEG

This example demonstrates that PEG and different amounts (%) of PEG canbe used for improving callus production. Corn embryos from corn earswere isolated manually as described elsewhere in the present disclosureand pooled. The embryos were stored in either 1 ml of Lynx 2304, or 1 mlof Lynx 2304 medium containing different amounts of PEG 8000 (SigmaP-2139) at 6° C. for 4 days in dark. Four replicates of each treatmentwere performed. The embryos were subsequently cultured on callusinduction medium Lynx 1074 for about 2 weeks. See Table 17 for Lynx 1074and 2304 media compositions. As shown in Table 16, in the controltreatment (no storage), 100% of the embryos produced callus when theywere cultured directly on Lynx 2304 medium after isolation. A smallpercentage of embryos produced callus when they were stored in 1% or 5%of PEG8000. None of the embryos produced callus when they were stored ina medium without PEG (trt 2). 100% of the embryos stored in 10 or 20% ofPEG 8000 produced callus (trts 5 & 6).

TABLE 16 Effect of PEG on callus response in corn immature embryos.Treatment % PEG (V/V) Culture response and comments 1 Control (no 100%of the embryos produced storage, cultured embryogenic callus responseimmediately after isolation) 2 0 Embryos didn't produce embryogenicculture response, died during culture 3 1 10% embryos producedembryogenic culture response 4 5 20% embryos produced embryogenicculture response 5 10 100% embryos produced 6 20 embryogenic cultureresponse

TABLE 17 Media compositions used in the invention. Media Components/L(Suppliers) Lynx1074 Lynx2304* MS Basal Salts Phytotech M524 4.33 g 4.33g MS Vitamins (100X) (Phytotec M533) 10 mL 10 mL Thiamine HCL (SigmaT-3902) 0.5 mg 0.5 mg 2,4-D (Phyotech D295) 0.5 mg 0 Sucrose (PhytotechS391) 30 g 30 g Proline (Sigma P-5607) 1.38 g 1.38 g Casamino Acids(Difco DF0288-17) 0.5 g 0.5 g pH 5.8 Low EEO Agarose (Sigma A-6013) 0 0Phytagel (Sigma P-8169) 3.0 g 0 Picloram (Sigma P-5575) 2.2 mg 0Carbenicillin (Phytotech C346) 0 0 Acetosyringone (Aldrich, D134406) 0 0BAP (Sigma B-3408) 0 0 Silver Nitrate (Sigma S-6506) 3.4 mg 0 *Volumewas adjusted to 800 ml and medium is stored after filter sterilization(FS) without pH adjustment. Prior to use, PEG and all other additives indescribed in Examples were added and volume/pH was adjusted prior to FS.

Example 10

This example demonstrates the use of PEG of different molecular weightsfor improving competency of plant cells. Corn embryos from corn earswere isolated manually as described elsewhere in the present disclosureand pooled, then stored in either 1 ml of Lynx 2304, or 1 ml of Lynx2304 medium containing 20% PEG of different molecular weight for 4 daysat 6° C. in dark. They were subsequently cultured on Lynx 1074 for 2weeks to observe callus response. As shown in Table 18, In the controltreatment (no storage), 100% of the embryos produced callus when theywere cultured directly on Lynx 2304 medium after isolation. No calluswas formed by embryos that were not stored in the absence of PEG, whilegenerally all embryos produced callus when contacted with PEG.Furthermore, the callus formation increased as the molecular weight ofPEG increased from 200 to 8000.

TABLE 18 Effect of various PEG species on callus response in cornimmature embryos. Ave Treat- Sigma Mol. m ment Cat# Wt osm/l Cultureresponse and comments A0 Control N/A 100% embryos produced embryogenic(No storage) callus A1 No PEG 171 Embryos didn't produce embryogenicculture response, died during culture A2 P- 200 1608 30% embryosproduced embryogenic 3015 culture response A3 P- 400 1116 50% embryosproduced embryogenic 3265 culture response A4 P- 950- 742 80% embryosproduced culture 3515 1050 response; good embryogenic callus A5 P- 1450626 Excellent embryogenic callus formation. 5402 Treatments wereundistinguishable from A6 P- 3350 586 each other 4338 A7 P- 8000 5322139

Example 11

This example illustrates improvement in transformation frequency of cornembryos contacted with PEG prior to transformation. Corn immatureembryos were isolated manually as described elsewhere in the presentdisclosure from inbred line LH 244 and divided into treatments. Embryosfrom one treatment were contacted with medium Lynx 2304 containing 20%WN PEG8000 for 10 min., then washed three times with sterile ddH₂O.Another treatment received no PEG treatment following embryo isolation.Both treatments were inoculated with Agrobacterium (OD 0.1 at 660 nm)for 5 minutes and subsequently cultured for selection and regenerationas described in U.S. Pat. No. 7,682,829.

As shown in Table 19, the embryos that were contacted with PEG producedhigher number of transgenic sectors and eventually higher number oftransgenic plants, indicating that contacting corn embryos with PEGimproves their transformation competency.

TABLE 19 Transformation frequency of corn embryos treated with PEG. #Embryos GFP # transgenic to selection positive plants % Treatment mediumSectors produced TF No PEG Treatment; 80 24 8 10 Agro OD = 0.1; 5 mininoculation PEG Treatment; 80 33 14 17.5 Agro OD = 0.1; 5 mininoculation

Example 12

This example demonstrates that competency of plant cells can be improvedby contacting the plant cells with a medium containing PEG and growthregulators such as auxins. Corn immature embryos were isolated manuallyas described elsewhere in the present disclosure from inbred line LH 244and stored in either 1 ml of Lynx 1854 (Table 20), containing 2.2 mg/Lpicloram 20% PEG 8000 (v/v) or Lynx 1854 without PEG 8000 at 4° C. for 6days in dark. The embryos were subsequently cultured on callus inductionmedium Lynx 1074 for about 2 weeks. FIG. 1 illustrates callus productiononly from the embryos that were stored in Lynx 1854 medium supplementedwith PEG and picloram (left panel), compared to embryos that were storedin only Lynx 1854 (right panel).

TABLE 20 Lynx 1854 medium composition Amount Ingredient Source per literMS Basal Salts Phytotech M524 4.33 g MS Vitamins Phytotech M533 103.1 mgThiamine HCL Sigma T-3902 0.5 mg 2,4-D Phytotech D295 0.5 mg SucrosePhytotech S391 30 g Proline Sigma P-5607 1.38 g Casamino Acids DifcoDF0288-17 0.5 g Polyethylene Glycol-MW 8000 Sigma P-2139 200 g PicloramSigma P-5575 2.2 mg pH to 5.8

Example 13

This example demonstrates improved transformation of corn embryos whenthey were stored in a medium containing PEG and one or more growthregulators. Corn immature embryos were isolated manually as describedelsewhere in the present disclosure from inbred line LH 244 and dividedinto treatments. Treatment 2 was stored in Lynx 1452 medium (Table 21)supplemented 20% (v/v) PEG 8000, 0.5 mg/l 2,4-D; 0.01 mg/L BAP (SigmaB-3408), while Treatment 3 was stored in Lynx 1452 supplemented with 20%(v/v) PEG, 0.5 mg/l 2,4-D; 2.2 mg/l Picloram. Treatments 2 and 3 werestored for 4 days at 6° C. in dark. For control (treatment 1), embryoswere not stored, nor contacted with PEG or any growth regulators priorto transformation. After storage the embryos were inoculated in 1 mLagro culture suspension for 5 minutes, the agro was removed, and thenthey were once again inoculated with 1 mL agro culture suspension foranother 5 minutes. Subsequent culturing and plant regeneration wasperformed according to the method described in U.S. Pat. No. 7,682,829.

As shown in Table 22, contacting corn embryos with a medium containingPEG and one or more growth regulators resulted in higher transformationfrequency.

TABLE 21 Lynx 1452 Medium Composition Amount Ingredient Source per literMS Basal Salts (Phytotech) Phytotech M524 4.33 g MS Vitamins (100X)(Phytotech) Phytotech M533 103.1 mg Thiamine HCL (Sigma) Sigma T-39020.5 mg Sucrose (Phytotech) Phytotech S391 30 g Proline (Sigma) SigmaP-5607 1.38 g Casamino Acids (Difco) Difco DF0288-17 0.5 g pH 5.8

TABLE 22 Transformation frequency of corn embryos contacted with PEG andvarious PGRs. # Embryos to selection # Events % Treatment Storage mediummedium produced TF 1 N/A 188 68 36.2 (Control) 2 Lynx 1452 w/ 269 15557.6 20% PEG 8000; 0.5 mg/l 2,4-D; 0.01 mg/L BAP 3 Lynx 1452 w/ 278 14451.8 20% PEG 8000; 0.5 mg/l 2,4-D; 2.2 mg/l Picloram

Example 14

This example demonstrates use of PEG alone or PEG in combination withgrowth regulators to improve competency of plant cells fortransformation at different temperatures. Corn immature embryos wereisolated manually as described elsewhere in the present disclosure frominbred line LH 244 and stored in either Lynx 2304 containing 20% (v/v)PEG 8000, or stored in Lynx 2304 containing 20% (v/v) PEG 8000 and 0.5mg/l 2,4-D+0.01 mg/l BAP. They were stored for 3 days at 4° C. or 23° C.in dark before being transformed according to the method described inU.S. Pat. No. 7,682,829. As shown in Table 23, corn embryos that werestored in a medium of Lynx 2304 plus PEG, or Lynx 2304 plus PEG andgrowth regulators, at 4° C. or 23° C. produced transgenic plants. Cornembryos that were stored in the medium containing PEG and growthregulators produced more transgenic plants than those stored in themedium containing no BAP. Corn embryos that were stored at 4° C.produced more transgenic plants than embryos that were stored at 23° C.

TABLE 23 Transformation frequency of corn embryos contacted with PEG andPGRs at various temperatures. # Embryos to selection # Events % StorageCondition medium produced TF Lynx 2304 + PEG @ 4 C. 203 45 22.2 (Totalof 4 replicates) Lynx 2304 + PEG + 2,4-D + 218 59 27.1 BAP @ 4 C. (Totalof 4 replicates) Lynx 2304 + PEG @ 23 C. 218 33 15.1 (Total of 4replicates) Lynx 2304 + PEG + 2,4-D + 228 47 20.6 BAP @ 23 C. (Total of4 eplicates)

Example 15

This example demonstrates use of PEG in combination with growthregulators to improve competency of plant cells for transformation whenembryos were stored for different periods of time. Corn immature embryoswere isolated manually as described elsewhere in the present disclosurefrom inbred line LH 244 and stored in Lynx 2304 containing 20% (v/v) PEG8000, 0.5 mg/l 2,4-D, 0.01 mg/l BAP for 3, 5, or 7 days at 4° C. indark. The embryos were then transformed according to the methoddescribed in U.S. Pat. No. 7,682,829 As shown in Table 24, corn embryosthat were stored either at 3, 5, or 7 produced transgenic plants. Cornembryos that were stored for 3 days produced more transgenic plants thanthose that were not stored in a medium containing PEG 8000 and growthregulators.

TABLE 24 Transformation frequency of corn embryos contacted with PEG andPGRs, stored for various durations of time. Embryos Transgenic StorageStorage to selection plants % period medium medium produced TF Nostorage N/A 393 173 44 3 d 20% PEG 8000; 421 203 48.2 0.5 mg/l 2,4-D;0.01 mg/l BAP 5 d 20% PEG 8000; 384 137 35.7 0.5 mg/l 2,4-D; 0.01 mg/lBAP 7 d 20% PEG 8000; 357 58 16.2 0.5 mg/l 2,4-D; 0.01 mg/l BAP

Example 16

This example demonstrates the use of PEG in improving competency ofplant cells that were isolated mechanically. Corn embryos were excisedmechanically as described in U.S. Pat. No. 7,560,611 and stored in Lynx2304 containing 20% (v/v) PEG 8000 for 2 days at 4° C. in dark. Theembryos were then transformed according to the method described in U.S.Pat. No. 7,682,829. As shown in Table 25, corn embryos that weremechanically excised were able to produce transgenic plants after twodays of storage in two different experiments.

TABLE 25 Transformation frequency of mechanically isolated corn embryostreated with PEG. # Embryos # Events % Expt to selection produced TF10194 133 36 27.1 10195 120 23 19.2

Example 17

This example shows that effective delivery of growth regulators to plantcells/tissues can be achieved by contacting the plant cells/tissues witha medium containing PEG and growth regulators such as a cytokinin priorto culturing the plant cells/tissues. Corn immature embryos wereisolated manually as described elsewhere in the present disclosure fromthe inbred line LH 244 and stored in either 1 ml of Lynx 2304 or 1 ml ofLynx 2304 containing 20% PEG 8000 (v/v), 5 mg/l BAP (v/v) at 4° C. for14 days in dark. The embryo explants were then plated on a germinationmedium after removal of the PEG. Just prior to plating the embryosstored in Lynx 2304 alone, 5 mg/l BAP (v/v) was added to storage tube.The embryos were subsequently cultured on germination medium Lynx 1607(Table 26) without any cytokinin for about one week. FIG. 2 illustratesenhanced trichome formation and swelling of the coleoptilar node fromthe embryos that were stored in Lynx 2304 medium supplemented with PEGand BAP (right panel) compared to the embryos that were stored withoutBAP (left panel).

TABLE 26 Lynx 1607 medium composition Ingredient Source Amount per literMS Basal Salts Phytotech M524 4.33 g MS Vitamins Phytotech M533 103.1 mgSucrose Phytotech S391 60 g Phytagar Gibco 10695-047 6 g CarbenicillinPhytotech C346 100 mg pH 5.8

Example 18

This example further demonstrates enhancement of competency of plantcells/tissues by contacting the plant cells/tissues with a mediumcontaining PEG and growth regulators such as a cytokinin. Corn immatureembryos were isolated manually as described elsewhere in the presentdisclosure from the inbred line LH 244 and stored in either 1 ml of Lynx2304 or 1 ml of Lynx 2304 containing 20% PEG 8000 (v/v) and 20 mg/l BAP(V/v) at 4° C. for 3 days in dark. The embryos were subsequentlycultured on callus induction medium Lynx 1074 for about 2 weeks. FIG. 3shows improved shoot formation from the embryos that were stored in Lynx2304 medium supplemented with PEG and BAP (top right, bottom rightpanels) compared to embryos that were stored without PEG or BAP (topleft, bottom left panels). Thus, this example also demonstratesenhanced, rapid regeneration of shoot and shoot-like structures fromembryogenic callus produced from explants that were contacted with PEGand a cytokinin for an effective period of time prior to culturing on anauxin-containing callus induction medium (Lynx 1074).

Example 19

This example demonstrates the effect of another commonly usedcryoprotectant osmoticum, glycerol, compared to PEG, in the storage ofembryos. Corn immature embryos were isolated manually as describedelsewhere in the present disclosure from the inbred line LH 244. Embryoswere transferred to eppendorff tubes containing lml of Lynx 1854, Lynx1854 with 20% glycerol and no PEG, or Lynx 1854 with both 20% glyceroland 20% PEG. There was one eppendorff tube each media. All of the threetubes were stored at 4 C for 6 days. After this incubation period thetubes were removed from the incubation and the entire contents from eachtube were transferred to 1074 semi-solid medium followed by spreadingout the immature embryos (20-25/plate). Culture response was scored 7days after incubating the embryos on 1074 under dark. As shown in Table27, culture response was only achieved form Treatment #1 with PEG alone.Either glycerol when present alone or in combination with PEG had anegative effect during storage as there was no culture response fromscutellar tissues.

TABLE 27 Effect of glycerol on culture response compared to PEGTreatment Culture Response Lynx 1854 (20% v/v PEG 8000) Yes Lynx 1854 +20% v/v glycerol, NO PEG No Lynx 1854 + 20% v/v glycerol and 20% v/v PEGNo

Example 20

Enhanced transformation of cotton cells contacted with PEG This exampledemonstrates enhanced transformation of dry cotton embryos that werecontacted with a composition containing PEG in sterile water. Cotton cv.DP393-0053 seeds were sanitized with 10% Clorox bleach for 10 minutes.They were then dried in a seed dryer overnight, achieving an averageinternal moisture content of 3.4%. Dry embryos were excised from the dryseeds by first processing the seed through a disc-style grinder, modelGP-140 (Modern Process Equipment Corp., Chicago, Ill.), and thenprocessed in an automated sieving and airflow separation device, likethe Clipper Eclipse 324 (Clipper Separation Technologies; A.T. FerrellCompany, Bluffton, Ind.) to remove a substantial portion of undesirableseed parts, such as seed coats, dust and other debris. The retained seedmaterial was immersed in liquid nitrogen until it stopped boiling (about30 seconds), and then processed once again through the disc-stylegrinder, before being additionally screened to isolate desired embryoexplant material from undesired material such as cotyledons, seed coatsand other debris, as described in U.S. Patent Application PublicationNo. 2008/0280361. Dry embryos were contacted for an hour with 20%PEG-4000 dissolved in sterile water. The embryos were rinsed for 4minutes in RO water, and then subjected to agrobacterium-mediatedtransformation according to the methods described in U.S. Pat. No.8,044,260. The embryos were regenerated on a spectinomycin selectionmedium. As shown in Table 28, embryos contacted with 20% PEG compositiongenerally showed enhanced transformation frequency, compared to embryoscontacted with INO medium (with no PEG). See Table 29 for spectinomycinselection medium composition for cotton.

TABLE 28 Transformation frequency of cotton cells contacted with PEG inwater compared to INO medium Treatment Initial Explants #R0 Plants TF %(stdev) INO 10000 140 1.4% (.26) 20% PEG4000 in 5000 120 2.4% (.38)water

TABLE 29 Spećtinomycin selection medium composition for cotton. AmountIngredient Source per liter Gamborg's B5 Medium Phytotech G398 3.21 gDextrose Fisher D16-3 20 g Calcium Gluconate Sigma G-4625 1.29 g Clearys3336 WP Carlin 10-032 0.03 g Agargel Sigma A-3301 4 g Carbenicillin 40mg/mL Phytotech C346 5 mL stock) Timentin (100 mg/mL Duchefa T0190 1 mLstock) Cefotaxime (50 mg/mL Midwest NDC0039-0019-10 4 mL stock)Spectinomycin (50 mg/mL Sigma S-4014 5 mL stock)

The invention claimed is:
 1. A method for improving competency of cornplant immature embryo for bacterial-mediated transformation comprising:contacting the corn plant immature embryo with an effective amount of apolyethylene glycol containing composition prior to transformation,wherein the effective amount of the polyethylene glycol is about 1% toabout 20% by volume.
 2. The method of claim 1, wherein the effectiveamount of the polyethylene glycol is about 20% by volume.
 3. The methodof claim 1, wherein the molecular weight of the polyethylene glycol isfrom about 200 to about
 10000. 4. The method of claim 3, whereinmolecular weight of the polyethylene glycol is from 4000 to
 8000. 5. Themethod of claim 1, wherein an effective amount of at least one plantgrowth regulator is provided with said polyethylene glycol containingcomposition.
 6. The method of claim 5, wherein the plant growthregulator comprises an auxin, cytokinin, or combination thereof.
 7. Themethod of claim 6, wherein the auxin is selected from the groupconsisting of IAA, 2,4-D, NAA, IBA, dicamba, or a combination thereof.8. The method of claim 6, wherein the amount of auxin is from about0.001 to about 30 mg/L.
 9. The method of claim 6, wherein the cytokininis selected from the group consisting of BAP, zeatin, kinetin, TDZ, or acombination thereof.
 10. The method of claim 6, wherein the amount ofcytokinin is from about 0.001 mg/L to about 30 mg/L.
 11. The method ofclaim 1, wherein the polyethylene glycol containing composition iscontacted with the corn plant immature embryo for about 30 minutes toabout 10 days.
 12. The method of claim 11, wherein the polyethyleneglycol containing composition is contacted with the corn plant immatureembryo for about 3 days to 7 days.
 13. The method of claim 11, whereinthe polyethylene glycol containing composition is contacted with thecorn plant immature embryo for about 30 minutes to 300 minutes.
 14. Themethod of claim 1, further comprising regenerating plant cells of thecorn plant immature embryo into whole plants.
 15. The method of claim 1,wherein the bacterial mediated transformation is Agrobacterium-mediated,Rhizobium-mediated, Sinorhizobium-mediated, Mesorhizobium-mediated, orBradyrhizobium-mediated transformation.
 16. The method of claim 1,further comprising a step of rinsing the corn plant immature embryocontacted with the polyethylene glycol in a non-PEG containingcomposition prior to transformation.
 17. The method of claim 1, furthercomprising the step of transforming the corn plant immature embryo witha heterologous DNA sequence via bacterial-mediated transformation.